Of central importance to insect growth, development and reproduction are the juvenile hormones (JH). Agonists of this hormone family are important alternatives for the control of medically important insect disease vectors. Yet, despite their widespread use, little is known about how JH acts at the molecular level. Much of the uncertainty stems from the lack of appropriate molecular tools, reliance on experimental models that may be many steps removed from the primary action of JH, and assumptions that the molecular targets of the hormone are identical in homeostasis, development and reproduction. A first step towards understanding JH action is identifying a molecular target. The black larval (bl) mutant of Manduca sexta displays a marked reduction in the hemolymph juvenile hormone binding protein (hJHBP) titer. We discovered that the bl fourth instar is extraordinarily sensitive to exogenous JH during the intermolt period; within 4 h of JH I application, the fat body exhibits a 4- to 6-fold increase in hJHBP mRNA. The rapidity and sensitivity of the response provides an attractive model for studying the molecular action(s) of JH on short-term homeostatic events. Towards that end, we have determined the genomic structure of the hJHBP gene and sequenced approximately 10 kb of its 5' and 3' flanking regions. The objectives of this proposal are: (1) determine the mechanism(s) by which JH increases levels of fat body hJHBP mRNA in bl larvae; (2) identify and sequence putative response elements using mobility shift assays and DNase I footprinting analysis; and (3) demonstrate the putative response elements are functional in cell transfection or in vitro transcription assays. Focusing on target gene regulation rather than a specific ligand-activated DNA binding protein allows strategic flexibility to explore new ideas about how the hormone acts at the molecular level. Although the gene-based approach may ultimately lead to a JH-activated DNA binding protein, it has the potential to identify all the key players, many of which may not interact with DNA or JH. The JH agonists are already used as growth regulators; expanding our knowledge about how JH acts will provide direction for designing new JH agonists and antagonists to control medically important insect disease vectors. Although the currently used agonists display no acute toxicity, they may interact with vertebrate gene regulatory elements and transcription factors, which in turn might lead to teratogenic abnormalities. Deciphering genetic regulatory elements common to insects and vertebrates is critical to expanding safe and more effective use of JH agonists.